“Biological control” or “biocontrol” is defined as pathogen suppression by the use of a second organism. Mechanisms of biological control are diverse. Biocontrol has long been thought to be safer for the environment and human health than synthetic pesticides (Cook et al. 1996; Benbrook et al., 1996). As bacterial biocontrol agents have reached the federal regulatory agencies for review, the agencies and the public have voiced concerns over the relatedness of some agents to human pathogens.
Bacillus species are widely used in agriculture as biocontrol agents of pathogens (e.g., oomycetes such as Pythium sp. and Phytopthera sp.) and insects (Handelsman et al. 1990; Silo-Suh et al. 1998; Shang et al. 1999). Bacillus thuringiensis is a biocontrol agent that produces insecticidal crystal toxin proteins, encoded by cry genes, that specifically kill insects including Lepidopterans, Dipterans, Coleopterans, Hymenopterans, and also kill nematodes. Methods for stabilizing and applying such toxins, or strains harboring the toxins, are known for a wide variety of field crop situations. Although distinct B. thuringiensis strains vary in target range and efficacy, the toxins required for biological control, and methods for preparing inocula for use in the field, are generally similar among strains.
Because B. thuringiensis is closely related genetically to food contaminant bacterium Bacillus cereus, concerns have been raised in the U.S. and Europe about its widespread use on food crops. Phylogenetic chromosomal marker studies show no taxonomic basis for separate species status for the two. While B. thuringiensis carries plasmids bearing the cry genes that encode insecticidal crystal toxins, B. cereus does not. On the other hand, B. cereus expresses chromosomally-encoded enterotoxin genes, the products of which are responsible for food-borne disease in humans, haemolysin BL (HBL), non-haemolytic enterotoxin (NHE) and cytotoxin K (CytK) (Beecher & MacMillan, 1991; Lund & Granum, 1996; Lund et al., 2000). Depending upon the strain, different toxins can be responsible for disease.
HBL and NHE are both three-component toxin complexes, which are restricted to the B. cereus group (From et al., 2005). HBL includes three component proteins, L2, L1 and B (Beecher & MacMillan, 1991), encoded by the genes hblC, hblD, and hblA, respectively, that are co-transcribed from the hblCDA operon (Heinrichs et al., 1993; Ryan et al., 1997; Lindback et al., 1999). NHE includes the proteins NheA, NheB and NheC, encoded by the nheABC operon (Granum et al., 1999).
Single component CytK belongs to the family of β-barrel pore-forming toxins (Fagerlund et al., 2008). Two cytK gene variants, cytK-1 and cytK-2, are known (Lund et al., 2000; Fagerlund et al., 2004). The original CytK-1 protein was isolated from a strain of B. cereus that caused three fatalities in a food poisoning outbreak (Lund et al., 2000). The CytK-2 version of the protein was subsequently identified from other strains of B. cereus (Fagerlund et al., 2004). This form is 89% identical to CytK-1 at the amino acid level and exhibits about 20% toxicity relative to CytK-1 toward human intestinal cells (Fagerlund et al., 2004).
A homolog of HBL has been discovered in the B. cereus group. Beecher and Wong (2000) showed that HBLa, isolated from a strain of B. cereus that also produced HBL, had similar toxicity as HBL and the homologous proteins could be interchanged. The 36 to 45 amino acids of the N-terminal sequence of the individual HBLa component proteins were reported in the Beecher and Wong study, but the gene sequences for HBLa were not known. However, an HBLa operon has been identified in the B. cereus UW85 partial genome sequence (D. Rasko, J. Ravel, J. Handelsman, unpublished). B. weihenstephanensis strain KBAB4 (Genbank accession CP000903) and B. cereus strain 03BB 108 (Genbank accession ABDM00000000) also contain HBLa sequences. The sequences disclosed in all cited Genbank accession numbers are incorporated herein by reference in their entirety as if set forth herein. The N-terminal sequences of the predicted HBLa proteins from UW85 are 100%, 69%, and 94% identical to the respective Ba, L1a, and L2a N-terminal sequences reported by Beecher and Wong (2000).
Some efforts to reduce or eliminate enterotoxin activity disrupted the components of the enterotoxin. U.S. Pat. No. 6,602,712 (Handelsman and Klimowicz; incorporated herein by reference as if set forth in its entirety) describes a Bacillus strain that exhibits reduced HBL enterotoxin activity. An alteration in the hblA gene of the hbl locus renders inactive the B component of the HBL protein. The other HBL components and other enterotoxin gene sequences were not disrupted. A corresponding component in the HBLa homolog may compensate for the lack of B component encoded by hblA.
When components NheB and NheC were eliminated from a B. cereus strain that lacked HBL and CytK, the strain lost haemolytic activity against erythrocytes from a variety of species (Fagerlund et al., 2008).
Prior attempts to eliminate the complete nhe operon in B. cereus and B. thuringiensis have failed (Ramarao & Lereclus, 2006; Fagerlund et al., 2008).
Many commercial B. thuringiensis strains, including subsp. kurstaki strain VBTS 2477, express such enterotoxin genes (Arnesen et al., 2008). The safety and public acceptance of B. thuringiensis on food crops would be enhanced by an enterotoxin-deficient B. thuringiensis strain that retains insecticidal activity but which does not produce an enterotoxin or its corresponding components. No B. thuringiensis strain is available that has reduced or zero levels of the enterotoxins or the functional components of the enterotoxins, including those components for NHE and HBL. Without the complete removal of these enterotoxins, the risk of toxicity remains.